Glucagon-like peptide (GLP) is an intestinal hormone secreted in human bodies, which is produced from the enzymatic lysis of proglucagon by intestinal protein hydrolase, wherein two kinds of glucagon-like peptides are generated, namely, GLP-1 and GLP-2. GLP-1 has two active forms, the GLP-1 (7-37) and the GLP-1 (7-36) with amidation, which possess with the same insulinotropic activity in vivo and are called Incretin or Insulinotropic peptide (Negar Sadrzadeh et al., Pharmaceutical Sciences, Vol. 96, 1925-1954 (2007)).
Exendin-4 is a polypeptide of 39 amino acids found in Heloderma horridurn venom, and shares similarities with many members of the GLP family, e.g., 53% homology with GLP-1 (7-36) (Eng J et al., J Biol Chem, 1992, 267: 7402-7405). As a strong GLP-1R agonist, Exendin-4 has been proved to be insulinotropic both in vivo and in vitro, and therefore can also be taken as an insulinotropic peptide. Amino acid sequences of GLP-1 (7-36) and Exendin-4 are as followings:
GLP-1 (7-36):His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Exendin-4:His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser
With Gly2 at the N-terminal, which is different from the Ala in GLP-1, Exendin-4 can effectively resist to the degradation of DPP IV. Furthermore, Exendin-4 comprises 9 amino acids (PSSGAPPPS) at the C-terminal that do not exist in GLP-1, making it unlikely to be digested by endonuclease, and is enhanced with the affinity to bind to GLP-1 receptors. Therefore, the blood sugar regulation ability of Exendin-4 is thousands of times more than that of GLP-1 in vivo. There are many analogues of Exendin-4, e.g. ZP10 a GLP-1 analogue with 5-lysine (KKKKK) added at the C-terminal of Exendin-4 (Petersen J S, Diabetologia, 2002, 45: A147); analogues with Lys20 substituted by Arg or His, Trp25 by Phe, and Met14 by Leu or Ile; as well as those with deletions at the C-terminal of Exendin-4, namely Exendin-4 (1-28) or Exendin-4 (1-30) and amidation forms thereof (Vilsbøll T, Diabetes Care, 2007, 30: 1608-1610).
Analogues of GLP-1 (7-36) and Exendin-4 are thus generated by the above modifications, comprising substitutions of one or more amino acids, or deletion or addition of the amino acid at the C-terminal.
GLP-1 (7-36) and Exendin-4, which have half-lives of only about 2 min and 2 hours in vivo, respectively, are reported in many patent publications and literatures to be fused to human blood albumin, the Fc fragment of human IgG or transferrin, to prolong the half-life (e.g., GLP-1-HSA, GLP-1-Fc, GLP-1-Transferrin, Exendin4-HAS, Exendin4-Fc, etc.; see U.S. Pat. No. 7,271,149; US2007/0036806A1; US 2007/0060512A1; US2006/0293232A1; and US2007/0161087A1). The above fusion proteins are mainly expressed and prepared using recombinant technology in host cells such as E. coli, yeast and mammalian cells (Xiaopu Yin et al., Protein Expression and Purification, 41 (2005), 259-265; Jin Zhou et al., Biotechnol Lett (2008) 30: 651-656; Fehmann H C et al., Peptides, 1994, 15(3): 453-456).
GLP-1 or Exendin-4 are reported to be modified by PEG in other patent publications and literatures, wherein they are conjugated mainly at the amino group of lysine, or COOH at the C-terminal (U.S. Pat. No. 6,284,727, U.S. Ser. No. 9/561,226), to prolong the half-life in vivo.
GLP-1 is also modified chemically at the aliphatic chain (e.g., Liraglutide) or at specific amino acids (Lys34) (e.g., CJC-1131) to be bound to albumn, so that it can be administrated once a day (with a half-life of 11-15 hours) (Kim, J G, Diabetes, 2003 March; 52(3): 751-759).
Besides of chemical solid-phase synthesis, GLP-1 and Exendin-4 are also reported to be expressed using recombinant DNA methods in literatures and patents, such as recombinant expression in E. coli (Xiaopu Yin et al., Protein Expression and Purification, 41 (2005)259-265); as well as in Pichia pastoris (Jin Zhou et al., Biotechnol Lett (2008) 30: 651-656). Furthermore, Exendin-4 is reported to be expressed in series as a dimer (Lina Yin et al., Protein & Peptide Letters, 2006, 13, 823-827).
GLP-1 or Exendin-4 mainly possesses with the following biological functions, including: (1) acting with pancreas islet β-cells to stimulate the synthesis and release of insulin; (2) inhibiting the release of glucagon and promoting the synthesis of glycogen; (3) increasing insulin sensitivity and sugar tolerance; (4) improving the proliferation and activity of pancreas islet (3-cell to reduce cell apoptosis; (5) reducing the rate of stomach evacuation; and (6) inhibiting appetite and energy uptake. Therefore, Exendin-4 (Trade name as Byetta) and Liraglutide have been granted to be polypeptide drugs for the treatment of diabete II, and relevant studies have been focused in the area.